1. Background Art
Lateral flow and flow-through technology have been used for diagnostic assays for almost twenty years. Lateral flow technology is currently dominant because lateral flow devices are easy to produce and the assay can be performed in a simple 2-step process that can be adapted for whole blood separation. This results in a simple device that can be used in the field as a rapid point-of-care diagnostic (Cole et al 1996 Tuberc. Lung. Dis. 77:363-368). However, multiple disease diagnosis using lateral flow technology is very difficult because of differences in lateral diffusion between samples and variation in flow rates between batches of the partitioning membrane. This means that antigen or antibody signal strengths may vary both within tests and between batches of tests, resulting in inconsistent results.
Existing flow-through diagnostic tests can be completed in less than two minutes compared with typical times of five to fifteen minutes for lateral flow tests. This advantage in speed however, is often at the expense of sensitivity. A further disadvantage is that higher volumes of sample are required to achieve the same sensitivity as lateral flow. This may be problematic in some situations. For example, the diagnosis of analytes (reagents) in whole blood requires the separation of plasma from whole blood cells. The higher volumes of whole blood required for this would quickly block the membranes in the flow-through format.
The basic principal of flow-through assays is well established. The tests are designed to determine the existence of, and in some cases, the quantity of, a predetermined analyte/reagent in a sample. Often the reagent will be a protein but other reagents can be tested for. If the assay is to test for the existence of a particular disease in a patient, the patient's body fluids may be tested for an antibody or other protein produced by the patient in response to the infection, or for a protein which is expressed by the bacterium or viral agent or the like causing the disease. In a typical flow through assay a liquid sample which is believed to contain the reagent is sucked into an absorbent pad via a membrane to which is bound a capture analyte which is known to bind to the reagent. The membrane is then typically washed with a buffer and a liquid containing a detection analyte which also binds to the reagent and which includes a tracer or marker which is detectable, is applied to the membrane. The detection analyte binds to the immobilised reagent bound to the membrane and can be seen or otherwise detected to indicate the presence of the reagent.
U.S. Pat. No. 4,246,339 discloses a test device for assaying liquid samples for the presence of a predetermined reagent. The device includes telescoping top and bottom members defining a liquid reservoir therebetween and resilient means for biasing the members in the open position. The top member defines a series of test wells each of which has a base defined by a microporous membrane with a capture analyte immobilised on the membrane surface. Absorbent means are located in the bottom member, spaced from the membrane in the open position but in contact therewith in the closed position. U.S. Pat. No. 4,246,339 discloses adding the test serum diluted with a buffer to a test well, and incubating the device at room temperature for ten minutes prior to depressing the cassette to the closed position to pass the sample through the membranes into the absorbent material, When the membranes are dry, the membrane is washed and then covered with a solution containing a detection analyte which binds to the immobilised reagent followed by a subsequent step in which a stain is applied.
It will be appreciated that the process described in U.S. Pat. No. 4,246,339, is a somewhat long drawn out, time consuming and tedious process and also lacks sensitivity.
A more recent flow through device is described in U.S. Pat. No. 5,185,127 which discloses an assay device including a filter stack and an enclosure having a base portion and a lid. The filter stack has a hydrophilic membrane having a capture analyte thereon, referred to in U.S. Pat. No. 5,185,127 as a binder. A hydrophobic membrane is located under the hydrophilic membrane and a pad of absorbent material is located under the hydrophobic membrane. The lid includes an upwardly extending rib which defines a recess having an insert therein. In use, a sample containing the reagent (referred to in U.S. Pat. No. 5,185,127 as the analyte) is placed in the well of the assay device at which time the reagent/analyte binds to the capture analyte/binder. Flow of the assay solution however, does not take place because the aqueous solution does not wet the hydrophobic membrane placed under the hydrophilic membrane in the filter stack. Thus as much time is necessary to complete the binding of the detection analyte to the reagent is allowed. When binding is judged to be complete, flow may be initiated by adding a wetting agent which wets the hydrophobic membrane. After which time the aqueous liquid flows into pad of absorbent material. The membrane may then be washed and treated with a detection analyte/tracer which may be an antibody which specifically binds to the analyte, the antibody having a label covertly conjugated thereto. Again the sensitivity of U.S. Pat. No. 5,185,127 is lacking and is not equivalent to that obtainable in lateral flow or ELISA formats.
2. General Information
As used herein the terms “derived from” or “derivative” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.
The embodiments of the invention described herein with respect to any single embodiment and, in particular, with respect to an apparatus or a method of assaying shall be taken to apply mutatis mutandis to any other embodiment of the invention described herein.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific examples described herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of immunocytochemistry such as for example immunogold labeling and proteomics. Such procedures are described, for example, in the following texts that are incorporated by reference:                Colloidal Gold—A New Perspective For Cytochemical Marking, Beesley J (1989), Royal Microscopical Society Handbook No 17. Oxford Science Publications. Oxford University Press. (Paperback);        An Introduction To Immunocytochemistry Current techniques and problems, Polak J and Van Noorden 5 (1984) Royal Microscopical Society Handbook No 11. Oxford Science Publications. Oxford University Press. (Paperback);        Immunocytochemistry—Modern Methods and Applications, Polak J and Van Noorden 5 (1986) (2nd ed), Butterworth Heinemann Oxford. (Hardback);        Techniques in Immunocytochemistry, Bullock G and Petrusz P (1982-1989) (4 volumes) Academic Press. Paperback); and        Colloidal Gold—Principles, Methods and Applications, Hayat M, (1989-1990) (3 volumes), Academic Press. (Hardback).        
All the references cited in this application are specifically incorporated by reference herein.
Because the prior art is not consistent in its terminology, for the avoidance of doubt and for the purpose of clarity, the following terms used in the specification below, are defined as follows. The term “reagent” or “target analyte” is used to refer to a macromolecule (eg. protein or enzyme) or fragment thereof, or the like which is to be detected by an assay. The term “capture analyte” is used to refer to a “capture” compound which is bound to a membrane and to which the reagent will bind. The term “detection analyte” is used to refer to an analyte which comprises a “detection” compound which will also bind to the reagent and also a “detectable” element. The detectable element is typically visually detected whether under visible light, or fluorescence.